The primary objective of Core B is to produce research grade rAAV vectors in a timely manner for use in the proposed rodent and dog studies of this U54 translational grant. The Vector Core will supply high-titer rAAV vectors of uniform quality to enhance experimental consistency and relieve the burden of large-scale vector production by the individual research laboratories. Moreover, our goal is to provide the vector as a uniform product minimizing lot-to-lot variation and maintaining consistent vector specific activity. This will be accomplished using an Ad-free transient transfection approach for small-scale mouse studies and a scalable producer cell line approach (that we developed) for high-titer rAAV preparations in support of the proposed GRMD dog studies. Innovations such as: (i) improved AAV helper plasmids for transient transfection production of serotypes 1, 6, and 8, (ii) universal purification method for these serotypes, and (iii) highthroughput cell line selection methods to rapidly identify high-titer producer cell lines (>5 x 104 vg per cell), will all be employed The Corning Cell Cube System will be used for high density mammalian cell culture (1010 cells) to achieve vector yields in excess of 1014 DNA vg. Density gradients and HPLC chromatographic methods will be used to isolate highly pure rAAV that is free of adenovirus and cellular protein contaminants, and vector quantity assessed using real-time quantitative PCR. Lastly, purified vector will be subjected to rigorous quality control assays (ie., sterility, replication competent AAV, replication competent adenovirus, endotoxin, total protein) to assure product consistency and uniformity.